Effects Of Direction-Induced Dedifferentiated Fat (d-DFAT) Cells On Repair Of Facial Nerve Defects In Rats

Facial nerve injury is one of the common but serious trauma complications in oral and maxillofacial surgery.Tumor invasion and improper operation of parotid gland may also lead to facial nerve injury and facial nerve paralysis.Peripheral facial paralysis caused by facial nerve injury is a serious disease that affects the facial motor function of patients(weakness of tympanic gills,inability of eyelids to close,etc.)and beauty(oblique mouth,disappearance of nasolabial sulcus,etc.).It will bring social,aesthetic and other psychological and mental disorders to patients and their families.In order to solve the above problems,the application of tissue engineering nerve grafts(TENGs)in tissue engineering and regeneration medicine(TE/RM)has attracted more and more interest in the repair of peripheral nerve defects.This kind of tissue engineering nerve graft includes: nerve catheter stent,supporting cells(seed cells),neurotrophic factor,extracellular matrix and cell adhesion molecule,etc.The structure and biological characteristics of the autograft are simulated,and an ideal regeneration microenvironment is established to better promote the regeneration of the nerve axons to the far end and to control the target organs.Clinically,adipose tissue can be obtained through repeated use of safe,minimally invasive liposuction,so it becomes an ideal source of seed cells.Mature adipocytes are the most abundant cell type in adipose tissue,accounting for more than90% of adipose tissue.Mature adipocytes isolated from adipose tissue can be dedifferentiated into fibroblasts-like cells in vitro(ceiling culture),called dedifferentiated fat cells(DFAT cells).Although DFAT cells and ASCs both are derived from adipose tissue,the cell populations of the two are different.DFAT cells are derived from mature adipocytes,while ASCs are derived from the stromal-vascular fraction of adipose tissue.SVF is composed of a heterogeneous group of cells,containing endothelial cells,non-characteristic stromal cells,blood cells and macrophages,etc.From the two sources,we can speculate that DFAT cells have higher homogeneity and purity than ASCs.DFAT cells are derived from mature adipocytes that account for more than 90% of adipose tissue.A large number of cellbases can be obtained in the early stage,which provides the possibility of rapid repair of facial nerve defects.Objective: This study intends to induce DFAT cells to be Schwann-like-like cells in vitro by in vitro cytokine induction,called direction-induced dedifferentiated fat(d-DFAT)cells,and further explore their roles in promoting facial nerve regeneration.Using d-DFAT cells combined with type I collagen gel and silicone catheter to construct a new type of tissue engineered nerve and evaluate the effect of repairing facial nerve defects.Method: 1.Isolate the primary cells of DFAT cells(ceiling culture method)and ASCs(differential centrifugation method),and carry out culture passage.Then observe whether DFAT cells have the same "stem cell characteristics" as ASCs by means of flow cell surface antigen identification,multi-directional differentiation ability and cell morphology.2.The second-generation DFAT cells and ASCs were induced to differentiate in vitro using cytokines,and the cell surface markers S-100βand GFAP of SCs were identified to determine whether they had the ability to differentiate into SCs.3.Seed cells(d-DFAT cells,d-ASCs,DFAT cells and ASCs)are mixed with type I collagen and co-cultured with silicone catheters to form TENGs.They were implanted into the SD rat facial nerve defect model.By comparing the vibrissae motor function of each group of rats,the electrophysiological evaluation of the regenerated facial nerve buccal branch nerve and the morphology and histology of the regenerated facial nerve buccal branch,the regenerative ability of each group on the facial nerve buccal branch defect of SD rats was judged.4.The 3D cell aggregates were constructed using d-DFAT cells and subjected to histological evaluation.Results: 1.The morphology of DFAT cells and ASCs are spindle-like fibroblast-like.DFAT cells can be differentiated into osteoblasts and adipocytes.Flow cell surface antigen identification showed positive expression of CD44 and CD90 but negative expression of CD45.2.DFAT cells differentiate under the action of nerve-inducing medium,and gradually change from fusiform,flat,multi-protruding stellate fibroblast-like cells to spindle-shaped,bipolar cell-like SCs.Immunecytochemistry identification confirmed that d-DFAT cells,like d-ASCs,express SCs surface markers S-100β and GFAP.3.Through the observation of SD rat’s vibrissae movement,the detection of neuroelectrophysiology and the evaluation of histological structure(myelin sheath,axon and glial cells).It can be seen that the recovery of facial nerve function and structure in the Schwann-like cell group(d-DFAT cell group and d-ASCs group)is superior to the stem cell group(DFAT cell group and ASCs group),but the overall situation is slightly inferior to the autograft group.4.The d-DFAT cells are cultured to form 3D d-DFAT cell aggregates.Through histological evaluation,we can find that the aggregates are composed of a large number of cells,and the density of the cells is higher and the arrangement is more uniform.And they contain a lot of collagen fibers and type I collagen,there are a lot of extracellular matrix between cells.Conclusion: 1.DFAT cells have the same stem cell characteristics as ASCs.2.DFAT cells can differentiate into Schwann-like cells.3.A 10 mm defect model of buccal branch of facial nerve was established in SD rats.TENGs containing seed cells(d-DFAT cells,d-ASCs,DFAT cells and ASCs)were used to repair the facial nerve buccal branch defects.Through evaluation of rat vibrissae movement,nerve electrophysiology,and morphology and histology of the regenerated facial buccal branch,it was found that d-DFAT cells as seed cells can well guide and support axon regeneration and promote the formation of myelin.Compared with stem cells as seed cells(DFAT cells,ASCs),d-DFAT cells have significant advantages in the repair of facial nerve buccal branch defects.However,compared with autologous nerve transplantation group,the ability of d-DFAT cells to regenerate facial nerve is still insufficient.4.Successfully constructed 3D d-DFAT cell aggregates.Through histological evaluation of the aggregates,it was found that the cells were evenly arranged,rich in collagen fibers and type I collagen,and there was a large amount of extracellular matrix between the cells.